Studies of animal tumor viruses such as SV40 have elucidated numerous mechanisms of both viral and cellular gene regulation. The first eukaryotic sequence-specific DNA binding protein to be isolated and characterized was the SV40 large T-ag. Early work on the SV40 T-ag revealed its remarkable multifunctional nature by its ability to mediate DNA replication, transcription and neoplastic tranformation. The studies of T-ag continue today with the recent high resolution X-ray structure of the T-ag dodecamer/ATPase which is responsible for DNA binding and initiation of viral DNA synthesis. The critical functions of the SV40 T-ag in controlling early viral and cellular gene regulation prompted our lab to launch a systematic analysis of the cellular machinery that works in conjunction with this viral trans-regulator to direct gene expression in animal cells. These studies have led to the discovery and biochemical characterization of over 75 transcription factors including sequence-specific enhancer/promoter recognition factors; core promoter/basal components of the pre-initiation RNA polymerase II complex; multi-subunit co-activators and chromatin remodeling co-factors. These biochemical studies have been combined with cell-based assays and gene targeting in the mouse to reveal an elaborate machinery which includes: cell-type specific and gene selective subunits of the core promoter recognition complex. In this grant renewal, we propose to continue our efforts to dissect the mechanisms by which the transcriptional apparatus operates to regulate gene expression in metazoan organisms. We will extend our effort to establish a highly purified in vitro transcription system using chromatinized DNA templates, focusing on uncovering the mechanisms of cell-type specific components of the core transcriptional machinery (i.e.TAFII105, TRF2, etc.). In addition to studying activation, we will renew our effort to study how viral and cellular proteins function in repression. Unlike previous grants, a significantly greater proportion of our work will be conducted in vivo (i.e. KO mice, immunofluorescence, FISH etc.). Finally, we will increase our effort in the area of structural analysis incorporating electron microscopy and single particle 3D reconstruction with X-ray crystallography to solve the 3D structure of large multi-subunit protein complexes. These studies should provide us with a comprehensive picture of how both viral and cellular genomes are expressed in a regulated fashion by the highly diversified transcriptional apparatus that has evolved in animal cells. Understanding the gene regulatory machinery has already provided the basis for developing various novel anti-cancer therapeutic strategies and we anticipate that as new target molecules are identified on the critical path leading to oncogenesis and disease, additional therapeutic interventions designed to intercede with gene regulatory mechanisms will emerge.